Antioxidants were purchased from Sigma-Aldrich Company. Study was conducted in two phases. Effect of supplementation of SOD, GPx, CAT and GSH to TEY buffer was observed on Labrador dog semen stored at 4ºC from 0-72 hrs. Different concentrations per antioxidant i.e. SOD (50-300 IU/ml), GPx (1.5-2.5 IU/ml), CAT (100-400 µg/ml) and GSH (2.5-10 µM/ml) were evaluated to look for an optimum dose.

All the procedures were approved by the CPCSEA, New Delhi vide F. No 25-19-2018-CPCSEA, dated 22/11/2018. Six Labrador dogs were maintained in individual pens. They were daily fed 500 g cooked feed twice daily and water provided ad libitum. Dogs were given regular exercise in the form of walking / running for one hour daily in the morning and evening. Dogs were dewormed and vaccinated for rabies, CDV, CAV2, CPV, CPI and CAV1. Semen was collected by message method twice a week.

Tris - citric acid-fructose buffer (Tris, 3.08 g; citric acid, 1.78 g; fructose, 1.25 g /100 ml, pH 7.2, gentamycin, 5 mg) was prepared and autoclaved at 15 lb for 15 min. Buffer was filtered through 0.2-micron membrane filter. Egg yolk and gentamycin at a concentration of 15 % and 50µg/ml were added to the buffer on the day of experiment, again filtered through 0.2-micron membrane filter and kept at 37°C (TEY extender).

Semen of dogs exhibiting >70 % motility was pooled for each trial and divided into required aliquots. To each aliquot SOD (50,100,200 and 300 IU/ml), GPX (1.5, 2.0, 2.5 µM/ml), CAT (100, 200, 300, 400 IU/ml) and GSH (2.5, 5.0, 7.5 and 10 µM/ml) were added and incubated at 37°C for 10 min. To each aliquot, extender was added and semen-extender mixture was centrifuged at 960 g for 3 min. Loose pellet was re-suspended in extender containing respective concentration of the antioxidant to get a final sperm concentration between 150-200 spermatozoa / ml. Sperm suspension was again equilibrated at 37^oC for 10 min and evaluated for motility, viability, membrane and acrosome integrity. Samples in tubes were shifted to a container containing warm water (37^oC) and placed in a cooling cabinet at 4ºC. Semen was analyzed for motility, viability and membrane integrity after every 24 hrs till 72 hrs of storage. Semen was also analyzed for acrosome integrity at 0, 72 hrs and lipid peroxidation at 72 hrs of storage.

Motility was evaluated by wet mount and track method. Viability was assessed by staining the spermatozoa with syber green-propidium iodide stain kit (Sigma). Hypoosmotic swelling test (HOST) was performed to analyze the integrity of sperm membrane (Jeyendran et al., 1984). One drop of semen incubated in 60 mosm HOS solution for 30 min was placed on a slide, covered with cover slip and examined under bright field microscope (Olympus) at 400 X for coiled tailed spermatozoa. A control was also run in PBS, pH 7.4. The number of coiled tailed spermatozoa in PBS was deducted from the number in hypo-osmotic solution and the resultant figure was taken as the HOS-reactive spermatozoa. For acrosome integrity, sperm smears prepared on clean glass slides were stained with spermac stain kit (Minitube) as per manufacturer’s instructions. About 200 spermatozoa were counted in different fields for all sperm attributes and percentage motility, viability, plasma membrane integrity and acrosome integrity were calculated.

Malondialdehyde concentration (end product of LPO) was estimated by the method of Buege et al., 1978). Briefly, semen was washed twice with PBS, pH 7.4 by centrifugation at 865 g for 5 min. Sperm pellet was suspended in 0.2 ml PBS, mixed with 0.1 ml of 150 mM Tris HCl (pH 7.1) and incubated at 37 °C for 20 min. After incubation, 0.5 ml 10% TCA and 1.0 ml 0.375% TBA were added and kept for 20 min in the boiling water bath. Thereafter, mixture was centrifuged for 15 min at 2500 g and absorbance of supernatant was taken at 532 nm. The molar extinction coefficient for MDA was calculated as below: MDA content (units) = OD × volume of assay mixture Volume of sample taken × coefficient extinction

Treatment of fresh semen with different concentrations of SOD and GPX at 37° for 10 min did not show significant difference in sperm attributes of freshly extended semen. However, treatment with 200 µg/ml catalase and 7.5µM/ ml GSH exhibited significantly higher values for sperm attributes in freshly extended semen compared to control and other tested doses (Table 1).

Preservation of semen in TEY extender supplemented with 100 and 200 IU SOD revealed significantly higher values of individual motility, viability and PMI at 24 hrs, 48 hrs and 72 hrs compared to control and 300 IU (Table 1). Values for motility, viability and PMI were non-significantly (p>0.05) higher in extender supplemented with 200 IU SOD than 100 IU during storage of semen at 4ºC from 24-72 hrs. Acrosome integrity was non-significantly different among the freshly extended - control and SOD treated semen. But it was significantly (p<0.05) higher in the presence of 200 IU SOD than control and other concentrations at 72 hrs of preservation. MDA production (µM/10⁹ spermatozoa) was non-significantly (p>0.05) higher in control than SOD treated semen samples at 72 hrs of preservation.

The values for motility, viability and plasma membrane integrity were significantly (p<0.05) high in TEY extender supplemented with 1.5 and 2.0 IU GPX / ml compared to control and other doses at 24 hrs of preservation (Table 2). However, these values remained significantly (p<0.05) high in TEY extender supplemented with 2.0 IU GPX / ml at 48 and 72 hrs of preservation. Acrosome integrity was also significantly (p<0.05) high in 2.0 IU GPX / ml supplementation at 72 hrs of preservation. Corresponding to sperm attributes, MDA concentration was significantly (p<0.05) less in the presence of 2.0 IU GPX / ml compared to control and other doses.

Supplementation of TEY extender with 200 µg/ml catalase resulted in significantly (p<0.05) higher motility, viability and plasma membrane integrity compared to control and other doses at 24, 48 and 72hrs of preservation (Table 3). Acrosome integrity also remained significantly (p<0.05) higher in 200 µg/ml dose compared to control and other doses. Surprisingly, MDA concentration in the presence of 200 µg/ml catalase was non-significantly (p>0.05) higher than control and 100 µg/ml and lower than 300 µg/ml.

Values for motility, viability and plasma membrane integrity were significantly (p<0.05) high in extender supplemented with 7.5 µM/ml GSH compared to control and other doses from 24 – 72 hrs of preservation (Table 4). Acrosome integrity was significantly (p<0.05) higher in 7.5 µM/ml GSH compared to control. But difference between different doses of GSH was non-significant (p>0.05). Similar to catalase supplementation, MDA concentration was non-significantly (p<0.05) high in the presence of 7.5 µM/ml GSH compared to control and other doses.

A gradual decline in sperm attributes was noticed with increase in time of preservation irrespective of the antioxidant treatment. This observation supported previous studies on preservation of chilled dog spermatozoa in extenders (Iguer-ouada and Verstegen, 2001; Thiangtum et al., 2012). Similar to our observations, higher viability compared to motility was observed during preservation of semen at 5°C (Thiangtum et al., 2012). Moreover, plasma membrane integrity and acrosome integrity were also higher than motility at 72 hrs of storage. Motility of Beagle dog spermatozoa was maintained up to 11 days by exchanging the old extender with fresh one (Verstegen et al., 2005). It was also suggested that viable spermatozoa may be reactivated by medium supplementation in vitro or in vivo when interaction with the vagino-utero-oviductal secretion occurs. But semen was preserved in the same extender up to 72 hrs of preservation in the present study. Therefore, decrease in sperm attributes with increasing storage period may be due to utilization of energy by the highly active spermatozoa and change in pH and osmolarity of extender. Reduction in motility with increasing storage period was attributed to the consumption of glucose, decrease in pH and metabolite accumulation (Rota et al., 1995; Verstegen et al., 2005). Many other factors may also influence the deterioration of chilled semen quality especially the lipid peroxidation due to oxidative stress during sperm storage in cooling condition.

It may be inferred that SOD, GPX, catalase and GSH acted in a dose dependent manner and 200 IU SOD, 2.0 IU GPX, 200 µg catalase and 7.5 µM GSH per ml semen were optimum doses for preservation of Labrador dog semen at 4^oC. Although motility, viability, plasma membrane integrity and acrosome integrity declined gradually in the presence of optimum dose of antioxidants, but still remained > 60% at 72 hrs of preservation. Contrary to our observations, extender containing 100, 400 and 1,600 U/ ml of CAT or SOD or the combination did not enhance sperm motility, viability and acrosomal integrity during storage at 5°C (Thiangtum et al., 2012). Comparable to our observations in dog, supplementing CAT and SOD and their combination to extender resulted in positive effect on chilled semen and during storage at 5°C in ram (Maxwell and Stojanov, 1996). Addition of CAT did not affect the maintenance of sperm motility during storage at 5°C in equine (Aurich et al., 1997; Ball et al., 2001). The addition of CAT to Tris-Lecithin extender did not improve even chilled dog semen (Beccaglia et al., 2009), but moderate benefit was reported when catalase at 100 U/ml was added to Tris-glucose egg yolk (Michael et al., 2009). The addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5°C for 24 h, although it did not affect the functionality of the sperm membranes (Câmara et al., 2011).

Addition of GSH at a concentration of 7.5 µM/ml was an optimum dose in improving sperm attributes compared to control during storage of Labrador dog semen at 4°C. Contrary to this observation, supplementation of the extender with 5 mM GSH improved the chilled and frozen-thaw semen quality (Angrimani et al., 2018; Monteiro et al., 2009). (Strezezek et al., 2009) reported that the physiologically normal GSH concentration in canine semen was closer to 5 mM, which is recommended for supplementation of semen extender/freezing solution. Variation in optimum dose of GSH had been reported in other species, as GSH @ 2.5 mM, 1 mM and 0.5 mM could preserve sperm quality of equine (de Oliveira et al., 2013), boar (Zhang et al., 2013) and bull (Tuncer et al., 2010) during storage of semen at 4°C, respectively.

Among the three concentrations of GPX tested, only 2 IU/ml supplementation to the extender could improve the sperm attributes in Labrador dogs. But, Del Prete et al. (2018) supplemented 15 IU of GPX and observed an improvement in DNA integrity during storage of semen at 4°C in fertile and hypo-fertile dogs.

It may be inferred from these studies that the different species respond variably to the antioxidants. Moreover, influence of environment including temperature, extenders and endogenous antioxidants may also be possible reasons behind variation. Different concentration of antioxidants should be evaluated for a species under investigation as per the conditions of particular lab, as optimum dose for one species may not be effective or may be toxic for the other species due to the change in physiological condition of semen extender. We also observed higher doses of antioxidants as toxic to dog spermatozoa. Addition of 400 mM and 10 mM GSH caused negative effects on ram (Câmara et al., 2011) and canine sperm (Ogata et al., 2015). In ram, survival of spermatozoa will increase when the dosage of SOD and CAT added to extender increases. However, CAT at dosage higher than 200 U/ml was toxic to ram spermatozoa (Maxwell and Stojanov, 1996).

There was no significant (p<0.05) difference in MDA concentration in control and antioxidant treated samples except GPX. Rather MDA concentration was nonsignificantly high in the presence of optimum catalase and GSH concentration. The addition of catalase in semen extenders resulted in significantly (p<0.0005) lower total ROS values compared with the controls in canine (Michael et al., 2009). GSH supplementation at a concentration of 5mM and 1mM inhibited lipid peroxidation levels in postthaw canine spermatozoa (Ogata et al., 2015) and beetal buck semen storage at 4°C (Sarangi et al., 2017). But, similar to our observations, MDA content of GSH treated groups was higher than that of the control group during storage at 4°C (Zhang et al., 2016). In spite of higher MDA content viz a viz lipid peroxidation in semen samples supplemented with optimum antioxidants concentration, the sperm attributes were higher compared to control and other doses.

In the dog, seminal fluid has been found to be deleterious for the in vitro preservation of sperm quality (Rota et al., 1995). It is also indicated that its presence before semen cryopreservation is also detrimental to sperm quality after thawing (Sirivaidyapong et al., 2001; Niżański, 2006). Therefore, seminal plasma was removed for processing, extension and preservation of semen at 4°C and main disadvantage of this method is the elimination of natural antioxidants from seminal plasma (Strzeżek et al., 2009). Semen has enzymatic (SOD, catalase and GPx) and non- enzymatic antioxidants (Michael et al., 2009). Canine spermatozoa contain antioxidant enzymes, such as SOD and GPX for protection against the cytotoxic effects of internally generated ROS (Strzeżek et al., 2009). However, the sparse cytoplasmic volume in the sperm cell midpiece limits its antioxidant capacity (Aitken et al., 1996). Moreover, intracellular antioxidant enzymes do not protect acrosomal and plasma membrane, so this must be protected by seminal plasma (Iwasaki and Gagnon 1992; Zini et al., 2002). ROS are produced by sperm in physiological conditions and play an important role on mammal sperm function (Rota et al., 1995; Beccaglia et al., 2009). ROS in low concentrations are necessary to achieve sperm fertilizing capacity, as they are involved in sperm kinetic, capacitation, hyper activation and fusion of the oocyte with the spermatozoa (Rota et al., 1995). Free radical concentration is controlled by the antioxidants present in seminal plasma and spermatozoa. The contribution of each antioxidant system varies among species and it is believed that there is a synergistic action between them (Michael et al., 2009). Antioxidants can act by inactivating free radicals (GSH, SOD, catalase, GPX and vitamin E) or repairing the oxidative stress lesions (Ferreira and Matsubara, 1997, ascorbate, glutathione reductase – GR and GPX). In the absence of seminal plasma, free radical concentration was maintained by supplemented antioxidants. Therefore, higher concentration of MDA and higher values of sperm attributes in the presence of optimum dose of antioxidants compared to control reveals that accomplished level of oxidative stress in preserved semen was not disproportionate and toxic to the spermatozoa. Otherwise, excessive production of free radical is responsible for alterations in semen quality, caused by decrease in sperm motility, as well as protein and DNA damage, resulting in cell apoptosis (Griveau and Le Lannou, 1997; Lucio et al., 2016). It may be revealed that supplementation of optimum dose of antioxidants to the extender fulfilled the function of seminal plasma antioxidants and resulted in boosted sperm attributes in preserved semen.